蔡珊珊臺灣大學：植物科學研究所劉育妏Liu, Yu-WenYu-WenLiu2007-11-272018-07-062007-11-272018-07-062007http://ntur.lib.ntu.edu.tw//handle/246246/57928從台東縣上里溫泉以及綠島朝日溫泉區域，分離篩選出六株嗜熱菌，經過初步之 16S rRNA gene 序列分析比對，可將這些菌株歸屬於 Chelatococcus 以及 Mesorhizobium 兩群。第一群均由上里溫泉分離出，包括 NTU-1599、NTU-1600 以及 NTU-1601 與 Chelatococcus asaccharovorans DSM 6462T 最相似。第一群分離菌株為好氧菌呈現黃色菌落，為具有移動性之格蘭氏陰性桿菌，最適生長溫度為 50 oC (生長溫度範圍介於 30-55)，且可生長於含鹽 6 % 之培養基，DNA 之 mol% G+C 組成為 64.2-64.7。另外， 第一群分離菌株為桿菌且不能利用 NTA 為碳源。這兩個主要特性可以很容易將它們與參考菌株 C. asaccharovorans 區分開來。根據 16S rRNA gene 序列比對相似度(96.55-96.83 %) 、 DNA-DNA 雜合反應結果 (8.5-17.5 %) 以及形態、生理生化等相關分析結果，初步判定第一群分離菌株為 Alphaproteobacteria 中的新菌種且可能為一新菌屬。 第二群皆由綠島朝日溫泉分離出，包括了 NTU-1636、NTU-1642 以及 NTU-1646 與Mesorhizobium loti DSM 2626T相似。第二群分離菌株為好氧菌具移動性，呈現黃色菌落，為具單鞭毛革蘭氏陰性桿菌，生長溫度範圍為30-55 oC (最適生長溫度為 40-50 oC)，可於15 % NaCl 之條件下生長。 另外，第二群的分離菌株與 M. loti皆可生長於不含氮的培養基中。第二群分離菌株之DNA 之 mol% G+C 組成為61.7-62.4。根據 DNA-DNA 雜合反應 (3.0-13.3 %) 和16S rRNA gene 序列分析結果(95.27-95.49 %) 以及生理生化等相關特性，可初步判定第二群分離菌株為 Mesorhizobium 屬之新種菌株。Six thermophilic bacteria were isolated from hot springs locating in the eastern region of Taiwan. The 16S rRNA gene sequence analysis indicated that these strains belonged to Alphaproteobacteria and could be classified into two groups: genus Chelatococcus and genus Mesorhizobium. Group I including strains NTU-1599, NTU-1600 and NTU-1601 isolated from hot springs in the Shangli area, Taitung county were closely related to Chelatococcus asaccharovorans DSM 6462T. Group I isolates were aerobic, yellow-pigmented, Gram-negative motile rods. The optimal growth temperature of isolates was 50 ℃ (tempterature range 30-55 ℃). Group I isolates could grow in the higher concentration of NaCl up to 6 % (w/v). The mol% G+C contents of the isolates were 64.2-64.7. The major differences between group I and the reference organisms were that group I isolates were rods and could not utilize NTA while the C. asaccharovorans was cocci and could utilize NTA (nitrilotriacetatic acid). According to the DNA-DNA homology (8.5-17.5 %), the 16S rRNA gene sequence analysis between the isolates and reference strain (96.55-96.83 %) as well as morphological, physiological, biochemical and chemical analyses indicated that group I isolates might be a novel genus within the Alphaproteobacteria. Group II including strains NTU-1636, NTU-1642 and NTU-1646 isolated from hot springs in the Lutao area, Taitung county were closely related to Mesorhizobium loti DSM 2626T. Those isolates were aerobic, motile, yellow-pigmented, Gram-negative rods with a single polar or subpolar flagellum. Growth temperature was between 30-55 ℃ (the optimal growth at 40-50 ℃). The isolates could grow in the medium containing NaCl up to 15 % (w/v). Both group II isolates and M. loti could proliferate in the nitrogen-free modified Thermus medium. The mol% G+C contents of the isolates were 61.7-62.4. Base on the DNA-DNA homology (3.0 -13.3 %), the 16S rRNA gene sequence analysis between the isolates and reference strain (95.27-95.49 %) as well as physiological, biochemical and chemical analyses indicated the isolates represented a novel member of the genus Mesorhizobium.口試委員會審定書 i 誌謝 ii 中文摘要 iii Abstract iv Abbreviations vi Contents vii List of Tables x List of Figures xi Introduction 1 1. Thermophilic bacteria 1 2. The application and interest on thermophiles 2 3. Thermophiles in Taiwan 3 4. Thermophilic Proteobacteria 4 5. Introductory of genus Chelatococcus 5 6. Introductory of genus Mesorhizobium 5 7. Aim of research 6 Materials and Methods 8 1. Materials 8 1.1 Source of bacterial strains 8 1.2 Chemicals and materials 8 1.3 Culture media 9 1.3.1 Modified Thermus medium 9 1.3.2 Modified Thermus basal medium 9 1.3.3 Nitrogen-free modified Thermus medium 9 1.4 Instruments 9 2. Methods 10 2.1 Sampling of bacterial strains 10 2.2 Isolation, purification and preservation 10 2.3 Morphological and ultrastructural characteristics 11 2.3.1 Morphology of colonies 11 2.3.2 Gram stain 11 2.3.3 Electron microscopic examination 12 2.3.4 Spreading motility 12 2.4 Physiological characteristics 12 2.4.1 Effect of temperature on growth 12 2.4.2 Effect of pH on growth 12 2.4.3 Effect of salt on growth 13 2.4.4 Single carbon sources utilization 13 2.5 Enzymatic characteristics and cellular properties 14 2.5.1 API-ZYM detection system 14 2.5.2 Starch hydrolysis 14 2.5.3 Casein hydrolysis 14 2.5.4 Gelatin hydrolysis 15 2.5.5 Elastin hydrolysis 15 2.5.6 Oxidase test 15 2.5.7 Catalase test 15 2.5.8 Lipids hydrolysis experiment 16 2.5.9 Chitin utilization 16 2.5.10 Chitosan utilization 16 2.5.11 Xylan utilization 17 2.5.12 PHB hydrolysis 17 2.5.13 Fatty acid profile 17 2.5.14 Antibiotic susceptibility test 18 2.5.15 Nitrogen fixation experiment 19 2.6 Genetic Properties 19 2.6.1. DNA extraction 19 2.6.2 16S rRNA gene ampliflication, sequence determination and phylogenetic analysis 20 2.6.3 DNA base composition 21 2.6.4 DNA-DNA hybridization 22 Results 24 1. Isolation of bacteria strains 24 2. Chelatococcus-like bacterial strains 24 2.1 16S rRNA gene sequence analysis 24 2.2. Morphological and ultrastructural characteristics 25 2.2.1. Colony morphology 25 2.2.2 Gram stain 25 2.2.3 Ultrastructural characteristics 25 2.2.4 Spreading motility 25 2.3 Physiologic characteristics 25 2.3.1 Effect of temperature on growth 25 2.3.2 Effect of pH on growth 26 2.3.3 Effect of salt on growth 26 2.3.4 Single carbon source utilization 26 2.4 Enzymatic characteristics and cellular properties 27 2.4.1 Enzymatic characteristics by the API-ZYM system 27 2.4.2 Other enzyme activities by the non-API-ZYM system 27 2.4.3 Fatty acid composition 27 2.4.4 Antibiotic susceptibility 27 2.5 Genetic Properties 28 2.5.1 DNA base composition 28 2.5.2 DNA-DNA hybridization 28 2.5.3 Phylogenetic analysis 28 3. Mesorhizobium spp. 29 3.1. 16S rRNA gene sequence determination 29 3.2. Morphological and ultrastructural characteristics 29 3.2.1. Colony morphology 29 3.2.2 Gram stain 29 3.2.3 Ultrastructural characteristics 29 3.2.4 Spreading motility 30 3.3 Physiological characteristics 30 3.3.1 Effect of temperature on growth 30 3.3.2 Effect of pH on growth 30 3.3.3 Effect of salt on growth 30 3.3.4 Single carbon source utilization 31 3.4 Enzymatic characteristics and cellular properties 31 3.4.1 Enzymatic characteristics by the API-ZYM system 31 3.4.2 Other enzyme activities by the non-API-ZYM system 31 3.4.3 Fatty acid composition 31 3.4.4 Antibiotic susceptibility 32 3.4.5 Nitrogen fixation test 32 3.5 Genetic Properties 32 3.5.1 DNA base composition 32 3.5.2 DNA-DNA hybridization 33 3.5.3 Phylogenetic analysis 33 Discussion 34 1. Sampling and isolation of bacterial strains 34 2. Characteristics of Chelatococcus-like isolates 35 2.1 Morphological characteristics 35 2.2 Physiological and biochemical characteristics 35 2.3 Fatty acid composition 37 2.4 Genetic characteristics and identification of group I isolates 37 2.5 Description of Chelatococcus-like isolates 38 3. Characteristics of Mesorhizobium spp. 39 3.1. Morphological characteristics 39 3.2. Physiological and biochemical characteristics 39 3.3. Fatty acid composition 40 3.4. Genetic characteristics and identification of group II isolates 41 3.5 Description of Mesorhizobium spp. 41 Conclusion 42 Reference 43 Tables and Figures 51 Appendix 922546893 bytesapplication/pdfen-US細菌台灣溫泉BacteriaTaiwanHot springotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/57928/1/ntu-96-R94b42001-1.pdf